This is a continuing proposal, for a comprehensive study of the biochemical and molecular basis of ornithine transcarbamylase (OTC) deficiency, an X-linked disorder, and the most common inherited defect of ureagenesis. Its focus is to increase understanding of structure- function relationships of the OTC enzyme (OTCase) and the biochemical and molecular basis of OTC deficiency. This study will employ bacterial and mammalian cell expression systems to investigate the effects of naturally occurring mutations on the synthesis, processing and catalytic function of human OTCase. To develop gene therapy approaches for certain mutants, we will investigate the assembly and biochemical properties of "chimeric" OTCase holoenzymes composed of wild type and mutant polypeptides. The crystal structures of the wild type human OTCase and several naturally occurring mutant enzymes will be refined and their catalytic mechanism will be investigated. Simultaneously, we will continue to expand the genotype-phenotype clinical database of OTC deficiency and will continue to provide updated information on the World Wide Web in a site dedicated to this disorder (www.peds.umn.edu/otc). The project has the following specific aims: (1) Study the enzymatic and physical properties of OTCase "chimeric" holoenzymes that contain both wild type and mutant polypeptides. (2) Perform expression studies of naturally occurring OTCase mutants in Chinese Hamster ovary (CHO) cells. (3) Complete the crystal structure determination of wild type and R277W and R277Q mutant human OTCase, determine the crystal structures of human OTCase complexed with various ligands and investigate the catalytic mechanism in solution. (4) Continue to expand our clinical database by characterizing molecular and biochemical defects in the OTC genes and enzymes of a large patient population with OTC deficiency. The ultimate goal is to help families with OTC deficiency with accurate diagnosis, reliable carrier testing and prenatal diagnosis and development of better treatment, especially gene therapy, through a better understanding of biochemistry and molecular biology of OTC deficiency.